AKT1 induces Nanog promoter in a SUMOylation-dependent manner in different pluripotent contexts

AKT/PKB is a kinase crucial for pluripotency maintenance in pluripotent stem cells. Multiple post-translational modifications modulate its activity. We have previously demonstrated that AKT1 induces the expression of the pluripotency transcription factor Nanog in a SUMOylation-dependent manner in mouse embryonic stem cells. Here, we studied different cellular contexts and main candidates that could mediate this induction. Our results strongly suggest the pluripotency transcription factors OCT4 and SOX2 are not essential mediators. Additionally, we concluded that this induction takes place in different pluripotent contexts but not in terminally differentiated cells. Finally, the cross-matching analysis of ESCs, iPSCs and MEFs transcriptomes and AKT1 phosphorylation targets provided new clues about possible factors that could be involved in the SUMOylation-dependent Nanog induction by AKT. Supplementary Information The online version contains supplementary material available at 10.1186/s13104-023-06598-3.


Additional Figures
summarizing their main features (SUMOylation capability [3] and effect on the Nanog promoter in ESCs [4]).SUMO is represented by yellow balloons, hyperactivity-inducing mutations are portrayed as spiny edges and the two lysines replaced by arginine in 2KR and E17K/2KR mutants are indicated by the red Xs.     3).mRNA (green) and/or protein (light blue) expression levels (log2 fold change) from multi-omic analysis of ESCs differentiating to epiblast-like cells.Data analysis was performed in the Stem Cell Atlas data-mining platform (http://www.stemcellatlas.org/)from publicly available transcriptomic and proteomic data [5].

Cell culture
The W4 mouse ESCs line was supplied by the Rockefeller University Core Facility (NY, USA) and the iPSCs line was previously generated and validated by our group [6].ESCs and iPSCs were routinely cultured in 0.1 % gelatin-coated dishes with ESC medium containing DMEM, 100 mM MEM NEAA, 2 mM Glutamax, 100 mg/ml streptomycin, 100 U/ml penicillin, 0.1 mM 2-mercaptoethanol, 15% fetal bovine serum (FBS, Gibco), LIF and the two-inhibitors (2i) cocktail consisting of 3 μM CHIR 99021 (Tocris) and 1 μM PD0325901 (Tocris).U-2 OS (ATCC HTB-96) were cultured in DMEM supplemented with 10% FBS (Internegocios), 100 mg/ml streptomycin and 100 U/ml penicillin (Gibco).All the cell lines were maintained at 37 °C in a 5 % CO2 (v/v) incubator and passed every two or three days.All experiments were performed in these conditions unless otherwise indicated.Mycoplasma contamination was regularly assessed in all cell lines by genomic DNA extraction followed by PCR analysis [7].

Transfection and luciferase activity assay
For the luciferase activity assay, cells were harvested and plated in a 24-well plate with the corresponding medium.ESCs and iPSCs were plated at a density of 36000 cells/well, and U-2 OS at 16000 cells/well.After 24 h, the culture medium was replaced according to the specified conditions of each experiment and cells were transfected with both 650 ng of either the empty vector or the corresponding AKT1 variant encoding vector [3] and 600 ng of the Nanog5P reporter.This luciferase reporter was kindly provided by Austin Cooney (Addgene plasmid # 16337) [8].PEI (Linear Polyethylenimine 25 kDa, Polysciences Inc.) was used for cell transfection with a DNA/PEI ratio of 1:3, in the case of ESCs and iPSCs, and 1:5 for U-2 OS and NIH/3T3 MEF cells.After overnight incubation, medium was replaced and cells were lysed and assayed for luciferase activity 24 or 48 h later (as indicated), using the Dual Luciferase kit (Promega) on a GloMax Multi Detection System (Promega).Total protein mass was measured by Bradford method and used for normalization in each transfection assay.Experiments were performed in triplicate and repeated at least three times.No substantial differences were detected in the proportion of transfected cells and the expression levels for the different AKT variants verified by immunofluorescence and Western blot [4].
For the analysis in Figure 3, a publicly available dataset of a genome-wide RNA-seq experiment containing paired transcriptomic data of ESCs, iPSCs, and MEFs was retrieved from the Stemformatics database (SRP046744) [1].Annotation, transformation, and normalization of this dataset were previously performed, and procedures are available at Stemformatics documentation (https://www.stemformatics.org/about)[17,18].All transcripts with FPKM above two times the detection threshold were retrieved from the ESCs, iPSCs, and MEFs groups.A comparison of all expressed genes, either shared or non-shared between the groups, were performed for the Venn diagram.Groups were numbered from I to VII depending on the number of genes contained.Then, a to-the-date updated AKT1 targets from the PhosphoSitePlus database were retrieved [24].Procedures of validation and data curation of these targets are available at PhosphoSitePlus documentation (https://www.phosphosite.org/staticCurationProcess.action).Subsequently, a cross-matching analysis between the list of AKT1 targets and the transcript groups identified was performed.Details and associated function of each gene were retrieved from the Uniprot database [25].
For Figure 3D and Figure S5, data analysis was performed in the Stem Cell Atlas data-mining platform from publicly available transcriptomic and proteomic data [5].Annotation, transformation, and normalization of this dataset were previously performed, and procedures are available at Stem Cell Atlas documentation (http://www.stemcellatlas.org/).

Statistical Analysis
Data was analyzed as previously described [4,[26][27][28].Significance between groups was analyzed by linear mixed models (LMM).Residuals fitted normal distribution and homogeneity of variance.Otherwise, transformation of data (log) was applied in some cases to meet both assumptions.Post-hoc multiple comparisons between means were assessed using the Tukey's HSD test.Experimental results were expressed as mean ± standard error of the mean (SEM) of at least three biological replicates.Differences were regarded as significant at least with a p-value of ≤0.05.The statistical analysis was performed using Infostat Software [29] or the gls package of RStudio.Specific analysis information is presented in each figure legend.

Figure S1 .
Figure S1.Nanog expression and promoter state in MEF and PSCs.Data analysis of Nanog expression and epigenetic marks on its promoter in ESCs, iPSCs and MEFs.Stemformatics data-mining platform was used to perform the analysis from publicly available data [1, 2].Nanog expression levels from RNA-seq (transcript) and LC-MS (protein) are shown in the upper panel; epigenetic marks within the Nanog promoter region from Histone ChIP-seq (H3K4me3 and H3K27me3, associated to active promoters and repressive marks, respectively) and from cytosine methylation by Bisulfite-sequencing are shown in the lower panel.

Figure S2 .
Figure S2.Summary of SUMOylatability and previous results of the AKT variants used.Schematic representations of the AKT1 variants used in our current and previous work and table summarizing their main features (SUMOylation capability [3] and effect on the Nanog promoter in ESCs [4]).SUMO is represented by yellow balloons, hyperactivity-inducing mutations are portrayed as spiny edges and the two lysines replaced by arginine in 2KR and E17K/2KR mutants are indicated by the red Xs.

Figure S3 .
Figure S3.NANOG is not detected in the U-2 OS cell line.Representative epifluorescence microscopy images of NANOG immunofluorescence (IF) of the tumoral osteosarcoma U-2 OS cell line, transfected with a vector encoding eGFP-NANOG.Transfection and IF were performed as previously described [4].Scale bar represents 10 µm.The images show that endogenous NANOG is not detected, since the nontransfected cell (left, yellow arrow), evidenced by DAPI staining, has no NANOG signal (right panel).As a control of the IF, cells were transfected with a vector encoding NANOG fused to the fluorescent protein eGFP (eGFP-NANOG).This fusion protein was detected both through detection of eGFP fluorescence (middle panel) and by IF against NANOG (right panel).

Figure S4 .
Figure S4.OCT4 and SOX2 are not detected in MEFs and U-2 OS cells.Western blot analysis of protein extracts of ESCs, NIH/3T3 MEFs and U-2 OS evaluating the presence of OCT4 and SOX2.GAPDH was revealed as loading control.The sample loaded in the 3 rd lane corresponds to a cell line that is not included in this work.

Figure S5 .
Figure S5.Downregulation of Oct4, Sox2 and Nanog by shRNA.ESCs were transfected with the pLKO.1puroderived vectors encoding shRNA targeting Oct4 (shOct4), Sox2 (shSox2), Nanog (shNanog) and eGFP (shGFP), as indicated below each bar.mRNA levels of each specific target, indicated at the top, were evaluated by RT-qPCR, normalized to the geometric mean of Gapdh and Pgk1 and referred to the control (shGFP).Bars represent the mean ± SEM of three independent experiments.Asterisks (*) indicate significant differences compared to the corresponding control condition (p < 0.05).

Figure S6 .
Figure S6.Multi-omic analysis of genes from Groups III to V in ESCs differentiating to epiblast-like cells (Related to Figure3).mRNA (green) and/or protein (light blue) expression levels (log2 fold change) from multi-omic analysis of ESCs differentiating to epiblast-like cells.Data analysis was performed in the Stem Cell Atlas data-mining platform (http://www.stemcellatlas.org/)from publicly available transcriptomic and proteomic data[5].
Full gene list of Group I from the cross-matching analysis of Figure3.